eliminating false-negative hits in ATP-luminescence

eliminating false-negative hits in ATP‑luminescence viability screens by use of an alternative phenotypic approach with acumen® Cellista

introduction

Cell viability and proliferation assays are a fundamental tool in the drug discovery process. They are used to evaluate both the potency of compounds, as well as their toxicity profiles for drug safety assessments. Many commonly used plate reader assays (e.g. ATP-luminescence measurements) assume a linear relationship between the assay signal, the number of cells and their viability. However, as we illustrate here, this assumption is not always justified: ATP-luminescence measurements can significantly overestimate toxicity and underestimate potency, leading to false-negative viability/ efficacy hits.

Here we show the development and implementation of an alternative no-wash high content (HCA) assay using acumen, which eliminates common problems associated with ATP-luminescence measurements. We compare the drug-responses of several well-characterised anti-cancer drugs on HeLa cells, measured by acumen and a commercial ATP-luminescence detection system, and highlight key differences between the measurements.

materials and methods

cell culture and drug additionAll plates are prepared as duplicates, one for imaging on the acumen Cellista and the other for ATP-luminescence determination.

day 1Seed 500 HeLa cells in 25 μL of medium into the wells of a 384-well plate and culture overnight.

day 2Prepare 2-fold serial dilutions of drugs in medium (2x concentrated), add 25 μL to each well and incubate for 48 hours.

day 4HCA assay: add 5 μL of stain “master mix” in PBS to each well, to give final dye concentrations of 10 μM Hoechst 33342, 1 μM calcein-AM and 1.5 μM propidium iodide. Incubate the plate for 90 minutes at room temperature and then image on the acumen Cellista to determine the cell number, % live cells and cell cycle phase distribution for each well.

ATP-luminescence assay: add 40 μL of CellTiter-Glo reagent (Promega) to each well, agitate the plate for 60 minutes at room temperature and then read the luminescence signal on a multimode plate reader.

data analysisAverage and normalise the well readouts for each drug concentration to the control wells. Plot drug-response curves of the normalised values and fit to sigmoidal curves to derive EC50 values.

reagent

unit price

unit size & stock concentration

cost per well

calcein-AM

£233

1 mL, 1 mM

1.12p

proidium iodide

£85

10 mL, 1.5 mM

1.12p

Hoechst33342

£82

5 mL, 20 mM

0.04p

CellTiter-Glo

£56

10 mL (sufficient for 1 x 384-well plate)

14.58p

results

phenotypic assay validation % live determination
Control or doxorubicin-treated cells were classified as either live or dead based on the ratio of the green (calcein-AM, FL2) to the red (propidium iodide, FL3) fluorescence intensity per cell. The percentage of live cells was calculated from the total number of live and dead cells.

cell cycle determinationacumen cell cycle classification gates were derived from the total FL1 fluorescence intensity histograms of untreated cells, and of cells treated with 3 μM etoposide for 48h (Figure 1). The predominant cell cycle response for each drug is summarised in Table 1.

comparison of drug‑responses across different assay formats

The drug-response curves (Figure 3) and EC50 values (Table 1) show that the agreement between the acumen Cellista cell number and % live cell readings, and the ATP luminescence readings varies widely between the compounds.

For doxorubicin, a classic cytotoxic agent, there is very close agreement between all three readouts.

For etoposide and cycloheximide there is less agreement, with the EC50 values for the acumen % live cell readout differing from the cell count by more than 1 log unit. The discrepancy between the two acumen readouts immediately highlights the cytostatic potency of these compounds and the cell cycle profiles confirm that etoposide is indeed a cell cycle inhibitor, whereas cycloheximide (a protein synthesis inhibitor) by elimination must work via a different mechanism of action. Furthermore, the acumen Cellista Tiff images (Figure 4) show an unusual enlarged morphological phenotype conclusions cannot be drawn from the ATP-luminescence measurements: The cycloheximide drug-response curves mirror the acumen cell count, which taken in isolation would falsely suggest a highly toxic compound. The etoposide drug-response curve falls in between the acumen cell count and % live readouts, probably because the antiproliferative action of the drug is accompanied by an increase in average cell size (and mitochondrial mass). With etoposide, the ATP luminescence measurements would also overestimate the toxicity of the compound.

With staurosporine, the acumen readouts show clear monotonic drug-response curves, with a cytostatic mechanism of action at lower drug concentrations, which becomes cytotoxic only at higher drug concentrations. The ATP measurements are more complicated, with a bi-phasic dose response showing the composite effect of cell health and number. In this example, the bi-phasic nature of the dose response curve hints at different mechanisms of action operating at different drug concentrations, however, the effect is quite subtle and could easily be missed. As with etoposide and cycloheximide, staurosporine would appear much more toxic at lower concentrations than it actually is.

about acumen

TTP Labtech’s acumen is a laser scanning imaging cytometer designed to provide single-shot, whole well, content-rich cytometric and image-based analysis. Its F-theta lens gives a uniform illumination across the field of view with a large focused depth of field, which enables high throughput, whole well image acquisition across a range of plate types. acumen enables a wide range of fluorescent reagents to be combined in
multicolour, multiplexed assays. Its easy-to-use, template-driven software offers an industry-proven route for quick adoption across a wide range of applications.

about cellista

Windows based Cellista software is powerful yet approachable. Cellista gives you the best of both worlds: application fl exibility combined with simplicity of operation. The software has been refined over many years and allows even the most novice users to rapidly become confi dent with the instrument.

acumen Cellista uses the principle of cytometry, rather than image analysis. This intelligent cytometric approach is quick and easy to learn, eliminating the requirement for specialised operators and endless ongoing training. Data output files are small, enabling acumen Cellista to be integrated into a screening workflow with little change to your existing infrastructure.